The revolution in molecular biology provided by DNA cloning and sequencing techniques, X-ray crystallography and NMR spectroscopy has offered unprecedented insights into the molecules that underlie the life process. However, in contrast to the dramatic rate of progress in sequencing and structural approaches, there remain major stumbling blocks in applying modern molecular knowledge fully, as many of the analytical techniques presently available remain remarkably similar to those used in the relatively early days of molecular biology and biochemistry.
For example, conventional gel electrophoresis and “blotting” techniques for determining the presence and amount of a given messenger RNA (mRNA) in a cell requires vast quantities of cells (˜109), and 2 days to complete. Even the most advanced DNA array chip techniques require ˜2×107 cells. Accordingly, advances in fields ranging from molecular medicine and basic cell biology to environmental toxicology are being hampered by the bottleneck generated by the sensitivity and speed of these conventional analytical techniques.
A growing literature of chemical force microscopy (CFM) has shown that a modified Atomic Force Microscope (AFM) can be tailored to measure the binding force of interactions ranging from single hydrogen bonds and single receptor-ligand interactions to single covalent bonds. For example, an early study showed the force required to break a single hydrogen bond to be on the order of 10 pN and subsequent work enabled the direct measurement of receptor/ligand interactions (˜50-250 pN) and DNA hybridization (˜65 pN-1.5 nN). CFM has also been utilized to study conformational changes such as the deformation of the polysaccharide dextran by an applied force and have elucidated the unfolding of the protein titan (˜100-300 pN). In addition to the above experiments performed with CFM, important advances have been made with optical tweezers. In particular, they have been used to study step-wise forces in biological motor motion and sub-pN polymer dynamics.
While the range of forces associated with many biochemical systems are well within the capability of AFM instrumentation to detect, there are severe limitations to the systems in which these devices can be used. For example, an AFM cantilever in solution does not have the temporal response characteristics needed to permit the binding and unbinding of biological ligands and their receptors to be followed reliably. Especially important are variation on the few μs timescale, characteristic of important classes of conformational changes in large biomolecules. High frequency response is also critical to following the stochastic nature of receptor ligand interaction. Most receptor-ligand pairs interact dynamically: binding, remaining engaged for times ranging from microseconds to seconds (depending on the exact receptor-ligand pair), and then releasing. The analysis of biomolecules is thus limited by both the vast quantities of materials required and the smearing in time inherent in even the most sensitive assays to date.
Perhaps even more significant is the substantial size of the equipment required for performing AFM/CFM, and the density limits imposed by optical detection of the probe motion. In addition, although the sensing mechanism is generally compact, even the so-called “lab on a chip” devices optical detectors are typically employed which require large, complicated support machinery, such as readers and sample preparation apparatus. These are not portable or easily reduced in size.
Third, optical tweezers employ diffraction-limited spots, hence the optical gradient forces generated are far too spatially-extended to permit direct manipulation of individual biomolecules under study. Instead, biofunctionalized dielectric beads typically having diameters in the range 0.1 to 1 μm, are used to adhere to the analytes. Accordingly, this technology is not readily scalable to nanometer dimensions or to large-scale integration.
Finally, all of the aforementioned techniques involve force sensors with active surface areas that are quite large compared to the molecular scale; hence it can be very difficult to achieve single-molecule sensing.
Accordingly, a need exists for a system and method for single molecule sensing in solution having higher sensitivity and temporal response with reduced overall size and active surface area.